The proposed research investigates events associated with the terminal stages of mammalian erythropoiesis. These events include nuclear condensation, nuclear extrusion and maturation of newly formed reticulocytes into biconcave erythrocytes. These studies will provide basic information about normal erythropoiesis, and give insights into a variety of erythroid diseases such as megaloblastic anemia, anemia secondary to chemotherapy and spherocytic anemia in which normal nuclear and/or cytoskeletal function is impaired. An in vitro model system of erythropoiesis employing erythroblasts from spleens of mice infected with the anemia-strain of Friend virus will be used. The first specific aim is to determine the role of the filamentous cytoskeleton and membrane skeleton in the enucleation of mammalian erythroblasts. The actin-containing constriction ring found in the region between the incipient reticulocyte and extruding nucleus will be isolated and analyzed by biochemical and immunological means. The role of the cytoskeleton in segregating spectrin to the incipient reticulocyte will be studied using inhibitors of actin filaments and microtubules. Changes in the levels of cytoskeletal proteins and expression of their respective genes during erythroblast maturation will be determined using Western blots, Northern blots and in situ hybridization. The second specific aim is to biochemically and morphologically characterize the maturation of reticulocytes into biconcave erythrocytes. Reticulocytes and extruded nuclei will be separated from nucleated erythroblasts and from each other. The intracellular organelles and plasma membrane will be analyzed in reticulocytes as they mature in vitro and correlated with changes in reticulocyte morphology. The third specific aim is to determine the mechanisms of nuclear condensation and identification of the signals responsible for initiating nuclear extrusion in mammalian erythroblasts. Changes in synthesis rate, phosphorylation state and accumulation of specific proteins will be quantitated in nuclei isolated from erythroblasts at early, middle and late stages of maturation using two dimensional gel electrophoresis. Isolated nuclei will be fractionated to determine if those proteins which show marked changes are associated with chromatin, the nuclear envelope/pore complex or the insoluble nuclear matrix. Antibodies will be raised against proteins identified as having a possible role in nuclear condensation or extrusion in order to determine the distribution of those proteins within the nucleus during the maturation of erythroblasts.